Lactoferrin is known as an iron-binding protein occurring in external secretions such as milk, saliva and the like. It has been known that lactoferrin bears an important function in dietetic view point to transfer iron and that lactoferrin possesses bacteriostatic effect, due to its iron-binding property, against pathogenic bacteria which have higher iron-requiring property. Thus lactoferrin is one of the important milk proteins as a nutrient and also as an infection defensive substance for human infants and calves along with immunoglobulins and lysozymes. Lactoferin occurs in milk in two forms, iron-binding and iron-free forms. Though colostrum contains relatively much lactoferrin, but normal milk contains only a minor amount, for example, 1 liter of cow's milk contains only 250 mg of lactoferrin.
In spite of the useful physiological functions of lactoferrin, it has been difficult to isolate and purify lactoferrin from milk in industrial scale because of its minor proportion in milk.
Many attempts have been made to develop a process for producing lactoferrin some of which are enumerated hereunder.
A process has been proposed wherein casein or whey fraction obtained by precipitation of cow's skim milk at isoelectric point of pH 4.6 is subjected to ammonium sulfate fractionation, subsequently a specific fraction obtained is subjected to column fractionation using several kinds of ion-exchangers to thereby purify bovine lactoferrin (see: M. L. Groves, J. Am. Chem. Soc., Vol. 82, p.p. 3345-3350, 1960; M. L. Groves, Biochem. Biophys. Acta., Vol. 100, p.p. 154-162, 1965).
It has been also proposed that in the process of purification of lactoperoxidase, rennet whey which is adjusted to pH 7.0 is subjected to adsorption with weakly acidic cation-exchanger, the substances adsorbed to said exchanger are desorbed with desorbing fluid, the resulted fluid is subjected to ammonium sulfate fractionation, then a specific fraction obtained is subjected to column fractionation with calcium phosphate or with weakly acidic cation-exchanger during which step lactoferrin contained as an contaminant is fractionated as a by-product (see: M. Morrison et al, J. Biol. Chem., Vol. 228, p.p. 767-776, 1957; W. G. Gordon et al, Bichem. Biophys. Acta., Vol. 60, p.p. 410-411, 1962).
A process has been proposed wherein human breast milk is subjected to ammonium sulfate fractionation, to the resulted supernatant fraction ferric ammonium sulfate is added and then the resulted fluid mixture is subjected to column fractionation with weakly acidic cation-exchanger to thereby purify lactoferrin (P. Querinjean et al, Uer. J. Biochem, Vol. 20, p.p. 420-425, 1971).
A process has been proposed wherein human breast milk is diluted three fold with water containing ferric ammonium sulfate and then the resulted fluid mixture is subjected to column fractionation with weakly acidic cation-exchanger to thereby purify lactoferrin (B. G. Johansson, Acta Chem. Scand., Vol. 23, p.p. 683-684, 1969).
Also affinity chromatography method utilizing fixed monoclonal anti bovine lactoferrin antibody has been known (see: Unexamined Japanese patent application Gazette No. 61(1986)-145200).
Conventional methods, however, are unsatisfactory as the methods for industrial mass production of lactoferrin due to their low efficiencies.
Moreover, the conventional methods may inevitably deteriorate a large quantity of raw milk-materials, since they involve addition of one or more of substances to the raw materials during their processes, for example, addition of ammonium sulfate for fractionation, addition of iron containing fluid for modifying lactoferrin to iron-binding form, and addition of pH adjusting fluid and so on. Furthermore, in purification of lactoferrin, conventional methods were not simple owing to utilization of several kinds of exchangers, utilization of various desorbing conditions in column fractionation, utilization of ammonium sulfate fractionation and so on.
Application of the affinity chromatography method to industrial production of lactoferrin involves preparation of a large quantity of the antibody which entails a cost problem. Furthermore, the conditions of stability of the antibody is severely confined, and in fact it is very difficult to apply the method to industrial scale production.
Therefore, it is an object of the present invention to provide a new and useful process for producing bovine lactoferrin.
It is another object of the present invention to provide an improved process for producing lactoferrin in industrial scale without resulting notable changes to the composition and quality of the raw materials and without complicated procedures.
It is a further object of the present invention to provide a process for industrial production of bovine lactoferrin in high purity.